Method for Treating Diabetes and Other Glucose Regulation Disorders Using Stem Cells

ABSTRACT

Various embodiments of the invention provide methods of treating diabetes and other glucose regulation disorders. In one embodiment, the method comprises removing L-cells from a donor, obtaining stem cells from a patient, and culturing the L-cells in the presence of the stem cells under conditions such that the stem cells differentiate into stem cell-derived L-cells (SCDLC). An amount of the SCDLC is introduced into the patient sufficient to cause a lowering of the patient&#39;s blood glucose level after ingestion of food. In another embodiment, the method comprises removing K-cells from a donor, obtaining stem cells from a patient, and culturing the K-cells in the presence of the stem cells under conditions such that the stem cells differentiate into stem cell-derived K-cells (SCDKC). An amount of the SCDKC is introduced into the patient sufficient to cause a lowering of the patient&#39;s blood glucose level after ingestion of food.

RELATED APPLICATIONS

This application claims the benefit of priority to Provisional U.S.Patent Application No. 61/342,029, entitled “METHOD FOR TREATINGDIABETES AND OTHER GLUCOSE REGULATION DISORDERS USING STEM CELLS”, filedApr. 7, 2010; the aforementioned priority application being herebyincorporated by reference for all purposes.

FIELD OF THE INVENTION

Embodiments of the present invention relate to the use of stem cells forthe treatment of disorders. More specifically embodiments of theinvention relate to the use of stem cells for the treatment of diabetesand other glucose regulation disorders.

BACKGROUND OF THE INVENTION

There are a number of disorders or conditions known as glucoseregulation disorders in which the body is not able to regulate bloodglucose within normal levels. Diabetes is one such disorder. Diabetes isa condition in which a person has a high blood glucose level as a resultof their body either not producing enough insulin or because the cellsof the body do not properly respond to insulin. The most common types ofdiabetes are: 1) Type 1 diabetes, which results from the body's failureto produce insulin; 2) Type 2 diabetes, which results from the body'sresistance to insulin; and 3) gestational diabetes, in which pregnantwomen who have never had diabetes before, have a high blood glucoselevel during pregnancy.

Diabetes is a significant global health issue, with at least 171 millionpeople worldwide suffering from the disease, about 2.8% of thepopulation. The most common form of diabetes is Type 2 diabetes, whichaffects 90 to 95% of the U.S. diabetic population. Diabetes is typicallytreated through lifestyle modifications such as consuming an appropriatediabetic diet and exercising more, and through medications such asinsulin. In spite of advances in treatment, there still remains a needfor more efficacious treatment regimens for diabetes.

In the past 15 years, many discoveries have been made in understandingstem cells. There have been proposals to use stem cells for treating awide variety of afflictions, including Parkinson's disease, spinal cordinjuries, amyotrophic lateral sclerosis, and multiple sclerosis.Described herein are novel methods for the use of stem cells for thetreatment of diabetes and other glucose regulation disorders.

SUMMARY OF THE INVENTION

Embodiments of the invention provide methods for treating diabetes andother glucose regulation disorders. Many embodiments involve the use ofstem cells which are induced to differentiate into mimics of a patient'sL-cells or K-cells, such that a release of incretins from thedifferentiated stem cells results in the lowering of the patient's bloodglucose level and improved control of the patient's blood glucoselevels. In one embodiment, the method comprises removing L-cells from adonor, obtaining stem cells from a patient, and culturing the L-cells inthe presence of the stem cells under conditions such that the stem cellsdifferentiate into stem cell-derived L-cells (SCDLC). An amount of theSCDLC is introduced into the patient sufficient to cause improvedcontrol of the patient's blood glucose including lowering of thepatient's blood glucose levels following ingestion of a meal to normalblood glucose levels (also referred to herein as euglycemic levels). Inanother embodiment, the method comprises removing K-cells from a donor,obtaining stem cells from a patient, and culturing the K-cells in thepresence of the stem cells under conditions such that the stem cellsdifferentiate into stem cell-derived K-cells (SCDKC). An amount of theSCDKC is introduced into the patient sufficient to cause improvedcontrol of the patient's blood glucose including lowering of thepatient's blood glucose levels following ingestion of a meal toeuglycemic levels.

Further details of these and other embodiments and aspects of theinvention are described more fully below.

DESCRIPTION OF PREFERRED EMBODIMENTS

There are a variety of gastro-intestinal hormones used for theregulation of glucose levels in the body. Among these hormones areinsulin, glucagon and incretins. Insulin is produced by the beta cellsof the islets of Langerhans of the pancreas. Insulin triggers cells totake up glucose, thereby reducing the level of glucose in the blood.Glucagon is produced by the alpha cells of the islets of Langerhans ofthe pancreases. Glucagon has the opposite effect of insulin on bloodglucose levels, i.e., glucagon stimulates the release of glucose byliver cells thereby causing an increase in blood glucose levels.

Incretins are gastrointestinal hormones which cause an increase in theamount of insulin secreted by the beta cells of the pancreas aftereating. This increase in insulin release occurs even before bloodglucose levels are elevated. Additionally, incretins inhibit glucagonrelease by the pancreas. Thus, administration of incretins presents apossible mechanism for treating diabetes and other glucose regulationdisorders since the incretins have the potential of both raising bloodinsulin levels and lowering blood glucagon levels, thereby leading to atwo-pronged approach for lowering blood glucose.

Two incretins are glucagon-like peptide-1 (GLP-1), which is produced byL-cells of the intestinal mucosa, and gastric inhibitory peptide (alsoknown as glucose-dependent insulinotropic pepide or GIP), which isproduced by K-cells of the intestinal mucosa. GLP-1 and GIP are bothrapidly inactivated by the enzyme dipeptidyl peptidase-4 (DPP-4).Because of this rapid inactivation, exogenous administration ofincretins by injection has not been a practical approach for loweringblood glucose and, hence, treating diabetes and other glucose regulationdisorders.

Embodiments of the present invention relate to methods for using stemcells for the treatment of diabetes and other glucose regulationdisorders such as impaired glucose regulation disorder. In preferredembodiments, the stem cells used are desirably obtained from the bonemarrow of a patient but may be obtained from any tissue in the body(e.g., skin, umbilical cord blood or tissue (e.g., Wharton's jelly),dental tissue, etc.). Embodiments of such methods are generallyapplicable to the treatment of any kind of diabetes, including Type Idiabetes, Type II diabetes, and gestational diabetes, as well as otherglucose regulation disorders. Moreover, although treatment of humans ispreferred, the method may be used to treat diabetes and other glucoseregulation disorders in any mammal, including monkey, cow, sheep, pig,goat, mouse, rat, dog, cat, rabbit, guinea pig, hamster and horse.

In one embodiment, the present invention is directed to a method oftreating diabetes and/or other glucose regulation disorders such asimpaired glucose regulation disorder. The method comprises obtainingstem cells from a patient, removing L-cells from a donor, culturing theL-cells in the presence of the stem cells under conditions such that thestem cells differentiate into stem cell-derived L-cells (SCDLC) (i.e.,an autologous L-cell mimic), and introducing the mixture of SCDLC anddonor L-cells into the intestinal sub-mucosa of the patient. The SCDLCwill then reseed (in the submucosa of the intestine or other location)as functioning L-cells capable of secreting GLP-1, which can then causelowering of the patient's blood glucose levels in response to the eatingof a meal containing carbohydrates or fats (or other incretinstimulating compounds), and the donor L-cells will be destroyed by thepatient's immune system. The cells can be introduced and placedendoscopically or by other minimally invasive means known in thegastrointestinal medical arts. The patient may be also given variousmedications or compounds to enhance or optimize seeding of the SCDLCcells.

In another embodiment, the present invention is directed to a method oftreating diabetes or other glucose regulation disorder, with the methodcomprising obtaining stem cells from a patient, removing K-cells from adonor, culturing the K-cells in the presence of the stem cells underconditions such that the stem cells differentiate into stem cell-derivedK-cells (SCDKC) (i.e., an autologous K-cell mimic), and introducing themixture of SCDKC and donor K-cells into the intestinal sub-mucosa of thepatient. The SCDKC will reseed as functioning K-cells capable ofsecreting GIP, which can then cause lowering of the patient's bloodglucose level upon exposure to incretin stimulating nutrients (e.g.,glucose, etc.), and the donor K-cells will be destroyed by the patient'simmune system. If desired, a patient may be treated with both SCDLC andSCDKC.

In use, either of these approaches can be configured to not only lowerthe patient's blood glucose levels upon eating of food (e.g.,carbohydrate, fatty acid, etc.) but also to lower it euglycemicconcentrations after a selected time period after eating food (e.g., 2hours or less) so as improve the patient's overall level of bloodglucose regulation. In particular embodiments, either of theseapproaches can be used to lower the patient's blood glucose to belowabout 150, 120 or 100 mg/dl within two hours or less of a mealcontaining carbohydrates (e.g., glucose, sucrose, etc.), fatty acids orother incretin stimulating food. Further, such approaches can beconfigured to improve the patient's glucose regulation so as to maintaintheir blood glucose concentration within euglycemic levels (e.g., 150 to80 mg/dl, or 120 to 80 mg/dl) for extended periods of time such as aday, week, month or year. Further still, the patient's long term bloodglucose control can be monitored using glycosolated hemoglobin or otherrelated analytes and monitoring means and the patient can then undergosubsequent reseedings with SCDLC and SCDKC cells as needed in improvethe patient's long term glucose regulation within euglycemic levels. Forexample, additional reseedings can be done if the patient is found tohave extended periods (e.g., weeks, etc.) of hyperglycemia.

The stem cells used in various embodiments of the invention can includepluripotent cells and multipotent cells. Examples of pluripotent cellsinclude embryonic cells as well as reprogrammed cells discussed below.Examples of mulitpotent cells include mesenchymal stem cells alsoreferred to as multi-potent stromal cells (both referred to as MSC's).Whatever the type, the stem cells may be from any appropriate source.For example, adult stem cells may be obtained from the patient's bonemarrow, muscle, adipose tissue various connective tissue and other formsof tissue. If the patient is pregnant, stem cells such as MSC's may beobtained from umbilical cord blood and/or umbilical cord tissue (e.g.,Wharton's jelly). Alternatively, amniotic stem cells can be obtainedfrom a pregnant patient. Stem cells such as MSC's may also be collectedfrom dental (e.g., the teeth) tissue including the developing tooth budof for example, third molar or other molar.

Yet another source of stem cells includes reprogrammed cells (e.g.,epithelial cells) which are given pluripotent capabilities. Such inducedpluripotent stem cells can be made, for example, by reprogramming adultcells with protein transcription factors, such as reprogramming adultskin cells using the transcription factors Oct3/4, Sox2, c-Myc and Klf4;or the transcription factors Oct4, Sox2, Nanog and Lin28 as well asvariants and other like factors.

Stem cells are typically sustained in an undifferentiated state byculture on a feeder layer of mouse embryonic fibroblasts with theinclusion of serum in the culture medium. In some embodiments, humanstem cells may be maintained on a feeder layer of mouse embryonicfibroblasts with the inclusion of basis fibroblast growth factor (bFGF)in the culture medium.

For use in the methods described herein, L-cells or K-cells from a donorare typically removed by biopsy, e.g., by a needle biopsy or otherbiopsy device. However, any applicable method may be used, such asremoval of a part of the intestine via surgery, in order to obtainL-cells or K-cells from the donor. The tissue sample containing theL-cells or K-cells is dispersed through the use of proteases or otherappropriate enzymes and/or mechanical dispersal. The L-cells or K-cellsmay also be separated/sorted from the removed tissue sample usingvarious cell sorting methods and equipment known in the art, forexample, such as flow cytometry sorting methods including FACS sortingmethods. Still other sorting methods are contemplated.

The L-cells or K-cells are typically added to the culture mediumcontaining the stem cells, although adding the stem cells to a mediumcontaining the L-cells or K-cells is also contemplated. The density ofthe stem cells and L-cells or K-cells, as well as the ratio of stemcells to L-cells or K-cells, may be adjusted for optimal results.Depending on the particular L-cell or K-cell, maintaining the cellco-culture under conditions used to maintain the stem cells may also beadequate to induce differentiation of the stem cells into stemcell-derived L-cells (SCDLC) or stem cell-derived K-cells (SCDKC). Withsome L-cells or K-cells, it may be desirable to remove serum orparticular growth factors (e.g., bFGF) from the culture medium beforedifferentiation of the stem cells into SCDLC or SLDKC occurs. In somecases, it may be desirable to add one or more growth factors to theculture medium to induce differentiation. These approaches can beadapted for the particular L-cells or K-cells chosen using the methodsdescribed below. Conditions found to produce differentiation can beoptimized and repeated for the same or similar stem cells and donorcells (e.g., K or L cells).

Depending on culture conditions used to maintain the stem cells, theymay be pluripotent, multipotent or oligopotent, with the type of stemcell being chosen so as to provide SCDLC or SCDKC with the desiredcharacteristics. Differentiation of stem cells into SCDLC or SCDKC istypically monitored by determining the ability of the cells to produceGLP-1 or GIP, respectively, and/or the disappearance of one or more stemcell markers. For example, one can monitor the disappearance of one ormore of alkaline phosphatase, alpha-fetoprotein (AFP), bonemorphogenetic protein-4, brachyury, cluster designation 30 (CD30),cripto (TDGF-1), GATA-4 gene, GCTM-2, genesis, germ cell nuclear factor,hepatocyte nuclear factor-4 (HNF-4), nestin, neuronal cell-adhesionmolecule (N-CAM), OCT4/POU5F1, Pax6, stage-specific embryonic antigen-3(SSEA-3), stage-specific embryonic antigen-4 (SSEA-4), stem cell factor(SCF or c-Kit ligand), telomerase, TRA-1-60, TRA-1-81, or vimentin. If,for example, pluripotent stem cells are introduced into a host then, dueto their pluripotent nature, it is possible that these cells willdifferentiate into many different types of cells, possibly causing ateratoma. Hence, it is important to insure that the SCDLC or SCDKC havelost sufficient pluripotent stem cell characteristics so as to not giverise to a teratoma in the patient.

As an additional, or alternate, criterion for determining when the stemcells have differentiated into SCDLC or SCDKC, one can monitor whetherone or more cell markers of the L-cell or K-cell have appeared on theSCDLC or SCDKC. Preferably, one can monitor for the presence of one ormore antigens of the L-cell or K-cell, such as one or more surfaceantigens.

As still another additional or alternate criterion for determining whenthe stem cells have differentiated into SCDLC or SCDKC, the cells can beexposed in vitro to the presence of glucose and/or a fatty acid or otherincretin stimulating nutrient compound and then the cells can bemonitored for minimum production of either GLP-1 or GIP (this can bedetermined on a batch level or individually using cell sorting methodssuch as FACs). In specific embodiments, only cells which produce aminimum level of GLP-1 or GIP (or other desired incretin) can then beintroduced into the patient. In use, this approach provides a functionaltest for assuring that only those cells producing a minimum level of thedesired incretin (e.g., GLP-1 or GIP) and thus capable of producingimprovement in the patient's blood glucose control are introduced intothe patient.

In still another related approach, the SCDLC or SCDKC cells can beconditioned in vitro to enhance the in vivo (e.g., in the patient)release of the desired incretin (e.g., GLP-1 or GIP) upon exposure toglucose, fatty acid(s) or other incretin stimulating nutrient compound.Such conditioning can include a variety of means. For example, the SCDLCor SCDKC cells can be repeatedly exposed in vitro to selectedconcentrations of glucose and/or a fatty acid or incretin stimulatingcompound. The cells can be monitored for production of either GLP-1 orGIP or other desired incretin and the concentrations of glucose, fattyacid(s) or other incretin stimulatory nutrient compound can be titrated(up or down) on each round of exposure to enhance the release of thedesired incretin. Experiments can be done to determine the optimalconcentration of glucose, fatty acid, etc., for enhancing the release ofthe desired incretin for a particular combination of patient stem cellsand donor cells (e.g., L-cell, K-cells). In one variant of thisapproach, conditioning can be done when the patient's stem cells anddonor cells are being co-cultured to differentiate the stem cells intothe desired SCDLC or SCDKC cells. Using such an approach, the stem cellsare exposed to cell signaling peptides or other compounds secreted bythe donor cells to stimulate production of the desired incretin. In thisway, the stem cells are conditioned to produce the desired incretin in alike manner to the donor cells. Repeated cycles of conditioning can beperformed using a particular regimen of glucose/fatty acidconcentrations. As an alternative or addition to this approach, duringone or more exposures to incretin stimulating compound, or co-culturingwith the donor cells, the stem cells can be exposed to a particularregimen of electrical stimulation (e.g., voltages in the range of about1 to 10 mv, or 10 to 100 mv, with smaller and larger rangescontemplated). Electrical stimulation can also be done as a separateconditioning step, before or after other conditioning steps (e.g.,co-culturing, exposure to incretin stimulating compounds, etc.). Theelectrical stimulation can be done using various voltage sourcesincluding programmable voltage sources known in the art. Use of thelatter can be configured to reproduce a particular voltage conditioningregimen for the production of multiple batches of SCDLC's or SLDKC's orother like cells.

CONCLUSION

The foregoing description of various embodiments of the invention hasbeen presented for purposes of illustration and description. It is notintended to limit the invention to the precise forms disclosed. Manymodifications, variations and refinements will be apparent topractitioners skilled in the art. For example, embodiments of theinvention can be readily adapted for stem cells not disclosed herein, orother cells having stem cell like properties. Additionally, embodimentsof the invention can be adapted for patients having disorders other thanblood glucose regulation disorders. Further, embodiments of theinvention can be readily adapted for pediatric and even neonatalapplications.

Elements, characteristics, or acts from one embodiment can be readilyrecombined or substituted with one or more elements, characteristics oracts from other embodiments to form numerous additional embodimentswithin the scope of the invention. Moreover, elements or acts that areshown or described as being combined with other elements or acts, can,in various embodiments, exist as standalone elements or acts. Hence, thescope of the present invention is not limited to the specifics of thedescribed embodiments, but is instead limited solely by the appendedclaims.

1-61. (canceled)
 62. A method for treating diabetes, the methodcomprising: obtaining gastrointestinal hormone producing cells from adonor; wherein said gastrointestinal hormone regulates glucose in thebody of a human; obtaining stem cells from a patient; culturing thegastrointestinal hormone producing cells in the presence of the stemcells under conditions such that the stem cells differentiate into stemcell-derived hormone producing-cells (SCDHPC); and introducing an amountof the SCDHPC into the patient sufficient to cause a lowering of thepatient's blood glucose level after the ingestion of food.
 63. Themethod of claim 62, wherein the gastrointestinal hormone is insulinglucagon or an incretin.
 64. The method of claim 62, wherein thegastrointestinal hormone producing cells are L-cells.
 65. The method ofclaim 62, wherein the gastrointestinal hormone producing cells areK-cells.
 66. The method of claim 62, wherein the method is used fortreating Type I diabetes.
 67. The method of claim 62, wherein the methodis used for treating Type II diabetes.
 68. The method of claim 62,wherein the stem cells are induced pluripotent stem cells.
 69. Themethod of claim 62, wherein the stem cells are pluripotent stem cells.70. The method of claim 62, wherein the stem cells are multipotent stemcells.
 71. The method of claim 62, wherein the stem cells aremesenchymal stem cells.
 72. The method of claim 62, wherein the stemcells are oligopotent stem cells.
 73. The method of claim 62, whereinthe SCDHPC do not express a pluripotent stem cell marker.
 74. The methodof claim 73, wherein the SCDHPC do not express the cell surface antigenSSEA3.
 75. The method of claim 73, wherein the SCDHPC do not express thecell surface antigen SSEA4.
 76. The method of claim 73, wherein theSCDHPC do not express the cell surface antigen Tra-1-60.
 77. The methodof claim 73 wherein the SCDKC do not express the cell surface antigenTra-1-81.
 78. The method of claim 62, wherein the SCDHPC do not expressa multipotent stem cell marker.
 79. The method of claim 62, wherein theSCDHPC do not express an oligopotent stem cell marker.
 80. The method ofclaim 62, wherein the SCDHPC express at least one cell surface antigenmimic of a cell surface antigen of the gastrointestinal hormoneproducing cells obtained from the donor.
 81. The method of claim 62,wherein the patient's glucose level is lowered below about 150 mg/dlwithin two hours after ingestion of food.
 82. The method of claim 62,wherein the food comprises a carbohydrate or a fat.
 83. A method fortreating diabetes, the method comprising: obtaining gastrointestinalhormone producing cells from a donor; wherein said gastrointestinalhormone regulates glucose in the body of a human; obtaining stem cellsfrom a patient; culturing the gastrointestinal hormone producing cellsin the presence of the stem cells under conditions such that the stemcells differentiate into stem cell-derived hormone producing-cells(SCDHPC); and introducing an amount of the SCDHPC into the patientsufficient to maintain the patient's blood glucose levels withineuglycemic levels for an extended period.
 84. The method of claim 83,wherein the gastrointestinal hormone producing cells are L-cells. 85.The method of claim 83, wherein the gastrointestinal hormone producingcells are K-cells.